Fields of the Invention
The present invention refers to a method for assessment of neural function by establishing an analysis module, especially to a method by which neurite outgrowth and function are analyzed fast and representatively. The method uses important markers related to neurite outgrowth in combination with optimized image analysis flow and program commands. Thereby the method can not only assess nerve growth and formation of neural network at early stage but also act as a high-content drug screening platform for quickly detecting the effect of drugs on neural function.
Descriptions of Related Art
With the progress of cancer therapies, survival rate of cancer patients is increased markedly. Recently, due to the extension of life span of cancer patients, side effects of chemotherapy drugs have drawn much more attention. Among the side effects of chemotherapy, neuropathy significantly affects patient's quality of life. Hence, the development of appropriate pre-clinical assay models and objective assessments of chemotherapy-associated neurotoxicity are thus the critical steps to test potential therapies for toxic reduction or prevention.
The high-content analysis (HCA) has been broadly applied in immunological, cardiovascular or neurological researches. The HCA together with high-throughput screening allows users to obtain fluorescence images of cells in a multi-well plate automatically. Thus researchers can efficiently conduct tests and acquire a variety of quantitative information. However, there are some problems while using molecular markers for assessing neuronal function in high-throughput screening. Most people choose one single marker or only one single parameter to indicate neuronal function. For example, Huang Y et al. used Neuronal Class III β-Tubulin in a high content neuron-based screening for assessing morphology of neurons (J Neurosci Methods. 2010 Jul. 15; 190(2):299-309). Harrill J A et al. applied a high-content analysis to assess neurite outgrowth of primarily rat cortical neurons. The microtubule-associated protein 2 (MAP2) labeled area (dendrites) is deducted from the βIII tubulin labeled area, and the rest area represents axons (Neurotoxicology. 2013 January; 34:61-73). Nieland T J et al. used MAP2, Psd95-Syn1, and Gphn-Syn1 to define synaptogenesis (PLoS One. 2014 Mar. 14; 9(3):e91744). The methods mentioned above assess neural function only by a single indicator, such as neurite outgrowth or synaptogenesis, which is not comprehensive. However, simultaneous multiparametric analysis for detecting neurite outgrowth and synaptogenesis requires more complicate, time-consuming processes and molecular markers.
Moreover, usage of cell model for drug screening also affects analyzing procedure and duration. For example, immortalized neuronal cell lines are easier to amplify and grown but suffered from several concerns. First, chemicals added for inducing differentiation may complicate the assessment of tested drug regarding to the unpredictable drug-drug interaction. In contrast, primarily cultured cells are differentiated naturally. Second, the primarily cultured neurons shared more characteristic with normal neurons in vivo. However, primarily cultured neurons are more difficult to obtain and maintain, to optimize the in vitro culture period is essential.
To avoid aforementioned weaknesses, the high-content screening strategies combined with appropriate cell model and the automated image acquisition and analyses are essential for the establishment of efficiency and sensitivity in vitro neurotoxic assays.